In conventional liquid chromatography, using high quality components, extra column band spreading is usually on the order, e.g., of about 50-100 .mu.l or less and thus generally is not significantly detrimental to the analysis. This holds true also for the case of high performance analytical columns. However, where the additional element of a post-column reactor or suppressor device is used between the column and detector, some greater amount of band spreading is introduced. In the case of high performance analytical columns, for example, those having a resolution efficiency of about 20,000 theoretical plates or greater, such additional band spreading can be detrimental in its effect on detection sensitivity, and/or as a primary cause of sample interferences. The present invention, in this respect, provides a practical and effective solution for controlling sample band spreading in practicing post column derivatization and suppression reactions in the field of liquid chromatography.
Specifically, among prior liquid chromatographic apparatus and method which may be significantly improved by the teachings of this invention is the apparatus used in conventional Ion Chromatography, e.g., as described by Small, "Applications of Ion Chromatography in Trace Analysis", Academic Press, New York, Chapter in "Trace Analysis", Editor James F. Lawrence (In Press); (see also, U.S. Pat. No. 3,920,397). Ion Chromatography is an analysis method which is based on a post-column suppression reaction. In this respect, liquid sample placed on an ion exchange analytical column is displaced by an electrolyte eluent or mobile phase, and elutes in a highly conductive eluent background solution which interferes with sensitive conductimetric detection of the sample ions. A "suppressor" column comprising a bed of ion exchange particles is thus placed between the analytical column and detector, and is used to selectively convert the electrolyte eluent to, e.g., deionized water or other weakly ionized background solution in which the sample ions can be sensitively detected. However, because of detrimental band spreading generally only relatively small suppressor columns are useful in Ion Chromatography, and these undesirably require frequent regeneration or replacement, as well as place other critical restrictions on the method (such as the usable strength of the eluent solution). In addition, even with these small suppressor columns, band spreading is nevertheless frequently on the order of, e.g., 300 .mu.l, or in an amount which can degrade the resolution efficiencies of high performance chromatographic columns.
The prior art has also developed a considerable variety of chemical reagents of a nature generally considered suitable for addition to liquid chromatographic effluent to enhance the detection sensitivity of eluted sample bands with reference to a specific detector, usually a photometer or fluorometer, but including other detectors; or to enhance their sensitivity with respect to interfering bands which overlap bands of interest. Various reagents and reagent reactions specifically contemplated as useful for liquid chromatography, for example, are described in some detail by Snyder et al., Introduction to Modern Liquid Chromatography, 2nd Ed. (1979) 740-746, herein incorporated by reference. Also incorporated herein by reference to illustrate this state of the art are the reagents and reagent reactions taught by Gfeller et al., Journal of Chromatography, 142 (1977), pp. 271-281; Frei et al. and Jupille, Journal of Chromatographic Science, Vol. 17, March, 1979, pp. 152-159 and 160-167, respectively; and Vance Nau et al., Analytical Chemistry, Vol. 51, No. 3, March 1979, pp. 424-428. In spite of the longtime availability of the above and like prior art teachings, however, reagent addition chemistry has only infrequently been applied to past liquid chromatographic practices. For example, it is stated:
"Although the use of such reaction techniques after column chromatographic separation has been known for more than a decade [ ] little has appeared in relation to modern HPLC. One reason is the many technical problems [ ] that still have to be solved" (Gfeller et al., p. 271). PA0 "The adaptation of reaction detection to modern LC columns requires careful attention to [ ] the design of equipment, because extra column effects can be serious. For these reasons reaction detectors have so far found rather limited use in modern LC." (Synder et al., p. 740).
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Among the above-referenced "extra column effects" is frequently detrimental band spreading or band broadening. For example, Frei et al., state: "the construction of proper reaction detectors comprises a constant struggle against band broadening"; and Jupille states, in respect to reagent addition methods, "a need for hardware modification (with attendant loss in flexibility); and [ ] a risk of band broadening due to post-column mixing volume resulting in loss of resolution."
Accordingly, it is a primary objective of this invention to provide liquid chromatographic apparatus and methods in which improved utility and flexibility are achieved in the practice and implementation of post column suppression and/or reagent addition reactions. It is particularly an object of the invention to provide such liquid chromatographic apparatus and method in which band spreading inherently produced by a post column suppressor or reactor device is acceptably controlled, and wherein the primary utility of producing an effective suppression or reagent addition reaction is simultaneously and effectively accomplished.